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1. DSP1 and DSP4 act synergistically in snRNA 3' end maturation and pollen growth

   https://doi.org/10.1104/pp.19.00231  

   Pu, Xuepiao ; Meng, Chunmei ; Wang, Weili ; Yang, Siyu ; Chen, Yuan ; Xie, Qingjun ; Yu, Bin ; Liu, Yunfeng ( January 2019 , Plant Physiology)

   mall nuclear RNAs (snRNAs) play essential roles in spliceosome assembly and splicing. Most snRNAs are transcribed by the DNA-dependent RNA polymerase II (Pol II) and require 3' end endonucleolytic cleavage. We have previously shown that the Arabidopsis (Arabidopsis thaliana) Defective in snRNA Processing 1 (DSP1) complex, composed of at least five subunits, is responsible for snRNA 3' maturation and is essential for plant development. Yet, it remains unclear how DSP1 complex subunits act together to process snRNAs. Here we show that DSP4, a member of the metallo-β-lactamase family, physically interacts with DSP1 through its β-Casp domain. Null dsp4-1 mutants have pleiotropic developmental defects, including impaired pollen development, and reduced pre-snRNA transcription and 3' maturation, resembling the phenotype of the dsp1-1 mutant. Interestingly, dsp1-1 dsp4-1 double mutants exhibit complete male sterility and reduced pre-snRNA transcription and 3' end maturation, unlike dsp1-1 or dsp4-1. In addition, Pol II occupancy at snRNA loci is lower in dsp1-1 dsp4-1 than in either single mutant. We also detected miscleaved pre-snRNAs in dsp1-1 dsp4-1, but not in dsp1-1 or dsp4-1. Taken together, these data reveal that DSP1 and DSP4 function is essential for pollen development, and that the two cooperatively promote pre-snRNA transcription and 3' end processing efficiency and accuracy. 

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2. Production of Class II MHC Proteins in Lentiviral Vector‐Transduced HEK‐293T Cells for Tetramer Staining Reagents

   https://doi.org/10.1002/cpz1.36  

   Willis, Richard A. ; Ramachandiran, Vasanthi ; Shires, John C. ; Bai, Ge ; Jeter, Kelly ; Bell, Donielle L. ; Han, Lixia ; Kazarian, Tamara ; Ugwu, Kyla C. ; Laur, Oskar ; et al ( February 2021 , Current Protocols)

   Class II major histocompatibility complex peptide (MHC‐IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self‐proteins. While the related Class I MHC/peptide (MHC‐Ip) multimers are usually produced from subunits expressed inE. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC‐IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC‐IIp proteins that uses stable lentiviral vector−transduced derivatives of HEK‐293T cells. The expression design includes allele‐specific peptide ligands tethered to the amino‐terminus of the MHC‐II β chain via a protease‐cleavable linker. Following cleavage of the linker, HLA‐DM is used to catalyze efficient peptide exchange, enabling high‐throughput production of many distinct MHC‐IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label‐free methods, native isoelectric focusing gel electrophoresis or matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC‐IIp complexes that are highly homogeneous and that form the basis for excellent MHC‐IIp multimer reagents. © 2021 Wiley Periodicals LLC.

   This article was corrected on 19 July 2022. See the end of the full text for details.

   Basic Protocol 1: Lentivirus production and expression line creation

   Support Protocol 1: Six‐well assay for estimation of production cell line yield

   Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His‐tags

   Basic Protocol 2: Cultures for production of Class II MHC proteins

   Basic Protocol 3: Purification of Class II MHC proteins by anti‐leucine zipper affinity chromatography

   Alternate Protocol 1: IMAC purification of His‐tagged Class II MHC

   Support Protocol 3: Protein concentration measurements and adjustments

   Support Protocol 4: Polishing purification by anion‐exchange chromatography

   Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

   Basic Protocol 4: Peptide exchange

   Basic Protocol 5: Analysis of peptide exchange by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry

   Alternate Protocol 2: Native isoelectric focusing to validate MHC‐II peptide loading

   Basic Protocol 6: Multimerization

   Basic Protocol 7: Staining cells with Class II MHC tetramers

    

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3. Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N 2 reduction

   https://doi.org/10.1073/pnas.1810211115  

   Raugei, Simone ; Seefeldt, Lance C. ; Hoffman, Brian M. ( October 2018 , Proceedings of the National Academy of Sciences)

   Recent spectroscopic, kinetic, photophysical, and thermodynamic measurements show activation of nitrogenase for N₂→ 2NH₃reduction involves the reductive elimination (re) of H₂from two [Fe–H–Fe] bridging hydrides bound to the catalytic [7Fe–9S–Mo–C–homocitrate] FeMo-cofactor (FeMo-co). These studies rationalize the Lowe–Thorneley kinetic scheme’s proposal of mechanistically obligatory formation of one H₂for each N₂reduced. They also provide an overall framework for understanding the mechanism of nitrogen fixation by nitrogenase. However, they directly pose fundamental questions addressed computationally here. We here report an extensive computational investigation of the structure and energetics of possible nitrogenase intermediates using structural models for the active site with a broad range in complexity, while evaluating a diverse set of density functional theory flavors. (i) This shows that to prevent spurious disruption of FeMo-co having accumulated 4[e⁻/H⁺] it is necessary to include: all residues (and water molecules) interacting directly with FeMo-co via specific H-bond interactions; nonspecific local electrostatic interactions; and steric confinement. (ii) These calculations indicate an important role of sulfide hemilability in the overall conversion ofE₀to a diazene-level intermediate. (iii) Perhaps most importantly, they explain (iiia) how the enzyme mechanistically couples exothermic H₂formation to endothermic cleavage of the N≡N triple bond in a nearly thermoneutralre/oxidative-addition equilibrium, (iiib) while preventing the “futile” generation of two H₂without N₂reduction: hydrideregenerates an H₂complex, but H₂is only lost when displaced by N₂, to form an end-on N₂complex that proceeds to a diazene-level intermediate.

    

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4. Reductive cleavage of N , N ′-di- tert -butylcarbodiimide generates tert -butylcyanamide ligands, (Me 3 CNCN) − , that bind potassium both end-on and side-on in the same single crystal

   https://doi.org/10.1107/S205698902000732X  

   Chung, Amanda B. ; Ziller, Joseph W. ; Evans, William J. ( July 2020 , Acta Crystallographica Section E Crystallographic Communications)

   null (Ed.)

   N , N ′-Di- tert -butylcarbodiimide, Me 3 CN=C=NCMe 3 , undergoes reductive cleavage in the presence of the Gd II complex, [K(18-crown-6) 2 ][Gd II (N R 2 ) 3 ] ( R = SiMe 3 ), to form a new type of ligand, the tert -butylcyanamide anion, (Me 3 CNCN) − . This new ligand can bind metals with one or two donor atoms as demonstrated by the isolation of a single crystal containing potassium salts of both end-on and side-on bound tert -butylcyanamide anions, (Me 3 CNCN) − . The crystal contains [K(18-crown-6)(H 2 O)][NCNCMe 3 - kN ], in which one ( t BuNCN) − anion is coordinated end-on to potassium ligated by 18-crown-6 and water, as well as [K(18-crown-6)][η 2 -NCNCMe 3 ], in which an 18-crown-6 potassium is coordinated side-on to the terminal N—C linkage. This single crystal also contains one equivalent of 1,3-di- tert -butyl urea, (C 9 H 20 N 2 O), which is involved in hydrogen bonding that may stabilize the whole assembly, namely, aqua( tert -butylcyanamidato)(1,4,7,10,13,16-hexaoxacyclooctadecane)potassium(I)–( tert -butylcyanamidato)(1,4,7,10,13,16-hexaoxacyclooctadecane)potassium(I)– N , N ′-di- tert -butylcarbodiimide (1/1/1) [K(C 5 H 9 N 2 )(C 12 H 24 O 6 )]·[K(C 5 H 9 N 2 )(C 12 H 24 O 6 )(H 2 O)]·C 9 H 20 N 2 . 

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5. Biogenesis of telomerase RNA from a protein-coding mRNA precursor

   https://doi.org/10.1073/pnas.2204636119  

   Logeswaran, Dhenugen ; Li, Yang ; Akhter, Khadiza ; Podlevsky, Joshua D. ; Olson, Tamara L. ; Forsberg, Katherine ; Chen, Julian J.-L. ( October 2022 , Proceedings of the National Academy of Sciences)

   Telomerase is a eukaryotic ribonucleoprotein (RNP) enzyme that adds DNA repeats onto chromosome ends to maintain genomic stability and confer cellular immortality in cancer and stem cells. The telomerase RNA (TER) component is essential for telomerase catalytic activity and provides the template for telomeric DNA synthesis. The biogenesis of TERs is extremely divergent across eukaryotic kingdoms, employing distinct types of transcription machinery and processing pathways. In ciliates and plants, TERs are transcribed by RNA polymerase III (Pol III), while animal and ascomycete fungal TERs are transcribed by RNA Pol II and share biogenesis pathways with small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA), respectively. Here, we report an unprecedented messenger RNA (mRNA)-derived biogenesis pathway for the 1,291 nucleotide TER from the basidiomycete fungus Ustilago maydis . The U. maydis TER ( Um TER) contains a 5′-monophosphate, distinct from the 5′ 2,2,7-trimethylguanosine (TMG) cap common to animal and ascomycete fungal TERs. The mature Um TER is processed from the 3′-untranslated region (3′-UTR) of a larger RNA precursor that possesses characteristics of mRNA including a 5′ 7-methyl-guanosine (m 7 G) cap, alternative splicing of introns, and a poly(A) tail. Moreover, this mRNA transcript encodes a protein called Early meiotic induction protein 1 (Emi1) that is conserved across dikaryotic fungi. A recombinant Um TER precursor expressed from an mRNA promoter is processed correctly to yield mature Um TER, confirming an mRNA-processing pathway for producing TER. Our findings expand the plethora of TER biogenesis mechanisms and demonstrate a pathway for producing a functional long noncoding RNA from a protein-coding mRNA precursor. 

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